Exploring the PDZ, DUF, and LIM Domains of Pdlim5 in Dendrite Branching.

The branched architecture of neuronal dendrites is a key factor in how neurons form ordered networks and discoveries continue to be made identifying proteins and protein-protein interactions that direct or execute the branching and extension of dendrites. Our prior work showed that the molecular scaffold Pdlim5 and delta-catenin, in conjunction, are two proteins that help regulate the branching and elongation of dendrites in cultured hippocampal neurons and do so through a phosphorylation-dependent mechanism triggered by upstream glutamate signaling. In this report we have focused on Pdlim5's multiple scaffolding domains and how each contributes to dendrite branching. The three identified regions within Pdlim5 are the PDZ, DUF, and a trio of LIM domains; however, unresolved is the intra-molecular conformation of Pdlim5 as well as which domains are essential to regulate dendritic branching. We address Pdlim5's structure and function by examining the role of each of the domains individually and using deletion mutants in the context of the full-length protein. Results using primary hippocampal neurons reveal that the Pdlim5 DUF domain plays a dominant role in increasing dendritic branching. Neither the PDZ domain nor the LIM domains alone support increased branching. The central role of the DUF domain was confirmed using deletion mutants in the context of full-length Pdlim5. Guided by molecular modeling, additional domain mapping studies showed that the C-terminal LIM domain forms a stable interaction with the N-terminal PDZ domain, and we identified key amino acid residues at the interface of each domain that are needed for this interaction. We posit that the central DUF domain of Pdlim5 may be subject to modulation in the context of the full-length protein by the intra-molecular interaction between the N-terminal PDZ and C-terminal LIM domains. Overall, our studies reveal a novel mechanism for the regulation of Pdlim5's function in the regulation of neuronal branching and highlight the critical role of the DUF domain in mediating these effects.

Brain Circuits Mediating Opposing Effects on Emotion and Pain.

The amygdala is important for processing emotion, including negative emotion such as anxiety and depression induced by chronic pain. Although remarkable progress has been achieved in recent years on amygdala regulation of both negative (fear) and positive (reward) behavioral responses, our current understanding is still limited regarding how the amygdala processes and integrates these negative and positive emotion responses within the amygdala circuits. In this study with optogenetic stimulation of specific brain circuits, we investigated how amygdala circuits regulate negative and positive emotion behaviors, using pain as an emotional assay in male rats. We report here that activation of the excitatory pathway from the parabrachial nucleus (PBN) that relays peripheral pain signals to the central nucleus of amygdala (CeA) is sufficient to cause behaviors of negative emotion including anxiety, depression, and aversion in normal rats. In strong contrast, activation of the excitatory pathway from basolateral amygdala (BLA) that conveys processed corticolimbic signals to CeA dramatically opposes these behaviors of negative emotion, reducing anxiety and depression, and induces behavior of reward. Surprisingly, activating the PBN-CeA pathway to simulate pain signals does not change pain sensitivity itself, but activating the BLA-CeA pathway inhibits basal and sensitized pain. These findings demonstrate that the pain signal conveyed through the PBN-CeA pathway is sufficient to drive negative emotion and that the corticolimbic signal via the BLA-CeA pathway counteracts the negative emotion, suggesting a top-down brain mechanism for cognitive control of negative emotion under stressful environmental conditions such as pain.SIGNIFICANCE STATEMENT It remains unclear how the amygdala circuits integrate both negative and positive emotional responses and the brain circuits that link peripheral pain to negative emotion are largely unknown. Using optogenetic stimulation, this study shows that the excitatory projection from the parabrachial nucleus to the central nucleus of amygdala (CeA) is sufficient to drive behaviors of negative emotion including anxiety, depression, and aversion in rats. Conversely, activation of the excitatory projection from basolateral amygdala to CeA counteracts each of these behaviors of negative emotion. Thus, this study identifies a brain pathway that mediates pain-driven negative emotion and a brain pathway that counteracts these emotion behaviors in a top-down mechanism for brain control of negative emotion.

A Chemical Chaperone Decouples TDP-43 Disordered Domain Phase Separation from Fibrillation.

Ribonucleoprotein (RNP) condensations through liquid-liquid phase separation play vital roles in the dynamic formation-dissolution of stress granules (SGs). These condensations are, however, usually assumed to be linked to pathologic fibrillation. Here, we show that physiologic condensation and pathologic fibrillation of RNPs are independent processes that can be unlinked with the chemical chaperone trimethylamine N-oxide (TMAO). Using the low-complexity disordered domain of the archetypical SG-protein TDP-43 as a model system, we show that TMAO enhances RNP liquid condensation yet inhibits protein fibrillation. Our results demonstrate effective decoupling of physiologic condensation from pathologic aggregation and suggest that selective targeting of protein fibrillation (without altering condensation) can be employed as a therapeutic strategy for RNP aggregation-associated degenerative disorders.

Inactivation of PNKP by mutant ATXN3 triggers apoptosis by activating the DNA damage-response pathway in SCA3.

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an untreatable autosomal dominant neurodegenerative disease, and the most common such inherited ataxia worldwide. The mutation in SCA3 is the expansion of a polymorphic CAG tri-nucleotide repeat sequence in the C-terminal coding region of the ATXN3 gene at chromosomal locus 14q32.1. The mutant ATXN3 protein encoding expanded glutamine (polyQ) sequences interacts with multiple proteins in vivo, and is deposited as aggregates in the SCA3 brain. A large body of literature suggests that the loss of function of the native ATNX3-interacting proteins that are deposited in the polyQ aggregates contributes to cellular toxicity, systemic neurodegeneration and the pathogenic mechanism in SCA3. Nonetheless, a significant understanding of the disease etiology of SCA3, the molecular mechanism by which the polyQ expansions in the mutant ATXN3 induce neurodegeneration in SCA3 has remained elusive. In the present study, we show that the essential DNA strand break repair enzyme PNKP (polynucleotide kinase 3'-phosphatase) interacts with, and is inactivated by, the mutant ATXN3, resulting in inefficient DNA repair, persistent accumulation of DNA damage/strand breaks, and subsequent chronic activation of the DNA damage-response ataxia telangiectasia-mutated (ATM) signaling pathway in SCA3. We report that persistent accumulation of DNA damage/strand breaks and chronic activation of the serine/threonine kinase ATM and the downstream p53 and protein kinase C-δ pro-apoptotic pathways trigger neuronal dysfunction and eventually neuronal death in SCA3. Either PNKP overexpression or pharmacological inhibition of ATM dramatically blocked mutant ATXN3-mediated cell death. Discovery of the mechanism by which mutant ATXN3 induces DNA damage and amplifies the pro-death signaling pathways provides a molecular basis for neurodegeneration due to PNKP inactivation in SCA3, and for the first time offers a possible approach to treatment.

Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus.

UNLABELLED: Severe fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genus Phlebovirus in the family Bunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-ß) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens. IMPORTANCE: SFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these "infectious" extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses.

PTP-PEST targets a novel tyrosine site in p120 catenin to control epithelial cell motility and Rho GTPase activity.

Tyrosine phosphorylation is implicated in regulating the adherens junction protein, p120 catenin (p120), however, the mechanisms are not well defined. Here, we show, using substrate trapping, that p120 is a direct target of the protein tyrosine phosphatase, PTP-PEST, in epithelial cells. Stable shRNA knockdown of PTP-PEST in colon carcinoma cells results in an increased cytosolic pool of p120 concomitant with its enhanced tyrosine phosphorylation and decreased association with E-cadherin. Consistent with this, PTP-PEST knockdown cells exhibit increased motility, enhanced Rac1 and decreased RhoA activity on a collagen substrate. Furthermore, p120 localization is enhanced at actin-rich protrusions and lamellipodia and has an increased association with the guanine nucleotide exchange factor, VAV2, and cortactin. Exchange factor activity of VAV2 is enhanced by PTP-PEST knockdown whereas overexpression of a VAV2 C-terminal domain or DH domain mutant blocks cell motility. Analysis of point mutations identified tyrosine 335 in the N-terminal domain of p120 as the site of PTP-PEST dephosphorylation. A Y335F mutant of p120 failed to induce the 'p120 phenotype', interact with VAV2, stimulate cell motility or activate Rac1. Together, these data suggest that PTP-PEST affects epithelial cell motility by controlling the distribution and phosphorylation of p120 and its availability to control Rho GTPase activity.

Amyloid-ß oligomers as a template for secondary amyloidosis in Alzheimer's disease.

Alzheimer's disease is a complex disease characterized by overlapping phenotypes with different neurodegenerative disorders. Oligomers are considered the most toxic species in amyloid pathologies. We examined human AD brain samples using an anti-oligomer antibody generated in our laboratory and detected potential hybrid oligomers composed of amyloid-ß, prion protein, α-synuclein, and TDP-43 phosphorylated at serines 409 and 410. These data and in vitro results suggest that Aß oligomer seeds act as a template for the aggregation of other proteins and generate an overlapping phenotype with other neuronal disorders. Furthermore, these results could explain why anti-amyloid-ß therapy has been unsuccessful.

Role of a Pdlim5:PalmD complex in directing dendrite morphology.

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching. We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that each proteins' effects are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.

A CK2 and SUMO-dependent, PML NB-involved regulatory mechanism controlling BLM ubiquitination and G-quadruplex resolution.

The Boom syndrome helicase (BLM) unwinds a variety of DNA structures such as Guanine (G)-quadruplex. Here we reveal a role of RNF111/Arkadia and its paralog ARKL1, as well as Promyelocytic Leukemia Nuclear Bodies (PML NBs), in the regulation of ubiquitination and control of BLM protein levels. RNF111 exhibits a non-canonical SUMO targeted E3 ligase (STUBL) activity targeting BLM ubiquitination in PML NBs. ARKL1 promotes RNF111 localization to PML NBs through SUMO-interacting motif (SIM) interaction with SUMOylated RNF111, which is regulated by casein kinase 2 (CK2) phosphorylation of ARKL1 at a serine residue near the ARKL1 SIM domain. Upregulated BLM in ARKL1 or RNF111-deficient cells leads to a decrease of G-quadruplex levels in the nucleus. These results demonstrate that a CK2- and RNF111-ARKL1-dependent regulation of BLM in PML NBs plays a critical role in controlling BLM protein levels for the regulation of G-quadruplex.

Role of a Pdlim5:PalmD complex in directing dendrite morphology.

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching (Baumert et al., J Cell Biol 2020). We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/ spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that effects of each protein are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.

p120-catenin subfamily members have distinct as well as shared effects on dendrite morphology during neuron development in vitro.

Dendritic arborization is essential for proper neuronal connectivity and function. Conversely, abnormal dendrite morphology is associated with several neurological pathologies like Alzheimer's disease and schizophrenia. Among major intrinsic mechanisms that determine the extent of the dendritic arbor is cytoskeletal remodeling. Here, we characterize and compare the impact of the four proteins involved in cytoskeletal remodeling-vertebrate members of the p120-catenin subfamily-on neuronal dendrite morphology. In relation to each of their own distributions, we find that p120-catenin and delta-catenin are expressed at relatively higher proportions in growth cones compared to ARVCF-catenin and p0071-catenin; ARVCF-catenin is expressed at relatively high proportions in the nucleus; and all catenins are expressed in dendritic processes and the soma. Through altering the expression of each p120-subfamily catenin in neurons, we find that exogenous expression of either p120-catenin or delta-catenin correlates with increased dendritic length and branching, whereas their respective depletion decreases dendritic length and branching. While increasing ARVCF-catenin expression also increases dendritic length and branching, decreasing expression has no grossly observable morphological effect. Finally, increasing p0071-catenin expression increases dendritic branching, but not length, while decreasing expression decreases dendritic length and branching. These distinct localization patterns and morphological effects during neuron development suggest that these catenins have both shared and distinct roles in the context of dendrite morphogenesis.

Sources of cell-to-cell variability in canonical nuclear factor-κB (NF-κB) signaling pathway inferred from single cell dynamic images.

The canonical nuclear factor-κB (NF-κB) signaling pathway controls a gene network important in the cellular inflammatory response. Upon activation, NF-κB/RelA is released from cytoplasmic inhibitors, from where it translocates into the nucleus, subsequently activating negative feedback loops producing either monophasic or damped oscillatory nucleo-cytoplasmic dynamics. Although the population behavior of the NF-κB pathway has been extensively modeled, the sources of cell-to-cell variability are not well understood. We describe an integrated experimental-computational analysis of NF-κB/RelA translocation in a validated cell model exhibiting monophasic dynamics. Quantitative measures of cellular geometry and total cytoplasmic concentration and translocated RelA amounts were used as priors in Bayesian inference to estimate biophysically realistic parameter values based on dynamic live cell imaging studies of enhanced GFP-tagged RelA in stable transfectants. Bayesian inference was performed on multiple cells simultaneously, assuming identical reaction rate parameters, whereas cellular geometry and initial and total NF-κB concentration-related parameters were cell-specific. A subpopulation of cells exhibiting distinct kinetic profiles was identified that corresponded to differences in the IκBα translation rate. We conclude that cellular geometry, initial and total NF-κB concentration, IκBα translation, and IκBα degradation rates account for distinct cell-to-cell differences in canonical NF-κB translocation dynamics.

Wnt/ß-catenin-mediated p53 suppression is indispensable for osteogenesis of mesenchymal progenitor cells.

The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues. The canonical Wnt pathway signals MPCs to commit to osteogenic differentiation at the expense of adipogenic fate. In contrast to ß-catenin, p53's anti-osteogenic function is much less understood. Both activities are thought to be achieved through targeting Runx2 and/or Osterix (Osx, Sp7) transcription. Precisely, how Osx activity is dictated by ß-catenin or p53 is not clarified and represents a knowledge gap that, until now, has largely been taken for granted. Using conditional lineage-tracing mice, we demonstrated that chondrocytes gave rise to a sizable fraction of MPCs, which served as progenitors of chondrocyte-derived osteoblasts (Chon-ob). Wnt/ß-catenin activity was only required at the stage of chondrocyte-derived mesenchymal progenitor (C-MPC) to Chon-ob differentiation. ß-catenin- C-MPCs lost osteogenic ability and favored adipogenesis. Mechanistically, we discovered that p53 activity was elevated in ß-catenin- MPCs including ß-catenin- C-MPCs and deleting p53 from the ß-catenin- MPCs fully restored osteogenesis. While high levels of p53 were present in the nuclei of ß-catenin- MPCs, Osx was confined to the cytoplasm, implying a mechanism that did not involve direct p53-Osx interaction. Furthermore, we found that p53's anti-osteogenic activity was dependent on its DNA-binding ability. Our findings identify chondrocytes as an additional source for MPCs and indicate that Wnt/ß-catenin discretely regulates chondrocyte to C-MPC and the subsequent C-MPC to osteoblast developments. Most of all we unveil a previously unrecognized functional link between ß-catenin and p53, placing p53's negative role in the context of Wnt/ß-catenin signaling-induced MPC osteogenic differentiation.

The Wnt/PCP formin Daam1 drives cell-cell adhesion during nephron development.

E-cadherin junctions facilitate assembly and disassembly of cell contacts that drive development and homeostasis of epithelial tissues. In this study, using Xenopus embryonic kidney and Madin-Darby canine kidney (MDCK) cells, we investigate the role of the Wnt/planar cell polarity (PCP) formin Daam1 (Dishevelled-associated activator of morphogenesis 1) in regulating E-cadherin-based intercellular adhesion. Using live imaging, we show that Daam1 localizes to newly formed cell contacts in the developing nephron. Furthermore, analyses of junctional filamentous actin (F-actin) upon Daam1 depletion indicate decreased microfilament localization and slowed turnover. We also show that Daam1 is necessary for efficient and timely localization of junctional E-cadherin, mediated by Daam1's formin homology domain 2 (FH2). Finally, we establish that Daam1 signaling promotes organized movement of renal cells. This study demonstrates that Daam1 formin junctional activity is critical for epithelial tissue organization.

Genome-wide CRISPR screen uncovers a synergistic effect of combining Haspin and Aurora kinase B inhibition.

Aurora kinases are a family of serine/threonine kinases vital for cell division. Because of the overexpression of Aurora kinases in a broad range of cancers and their important roles in mitosis, inhibitors targeting Aurora kinases have attracted attention in cancer therapy. VX-680 is an effective pan-Aurora kinase inhibitor; however, its clinical efficacy was not satisfying. In this study, we performed CRISPR/Cas9 screens to identify genes whose depletion shows synthetic lethality with VX-680. The top hit from these screens was GSG2 (also known as Haspin), a serine/threonine kinase that phosphorylates histone H3 at Thr-3 during mitosis. Moreover, both Haspin knockout and Haspin inhibitor-treated HCT116 cells were hypersensitive to VX-680. Furthermore, we showed that the synthetic lethal interaction between Haspin depletion and VX-680 was mediated by the inhibition of Haspin with Aurora kinase B (AURKB), but not with Aurora kinase A (AURKA). Strikingly, combined inhibition of Haspin and AURKB had a better efficacy than single-agent treatment in both head and neck squamous cell carcinoma and non-small cell lung cancer. Taken together, our findings have uncovered a synthetic lethal interaction between AURKB and Haspin, which provides a strong rationale for this combination therapy for cancer patients.

Region-Specific Transcriptional Control of Astrocyte Function Oversees Local Circuit Activities.

Astrocytes play essential roles in brain function by supporting synaptic connectivity and associated circuits. How these roles are regulated by transcription factors is unknown. Moreover, there is emerging evidence that astrocytes exhibit regional heterogeneity, and the mechanisms controlling this diversity remain nascent. Here, we show that conditional deletion of the transcription factor nuclear factor I-A (NFIA) in astrocytes in the adult brain results in region-specific alterations in morphology and physiology that are mediated by selective DNA binding. Disruptions in astrocyte function following loss of NFIA are most pronounced in the hippocampus, manifested by impaired interactions with neurons, coupled with diminution of learning and memory behaviors. These changes in hippocampal astrocytes did not affect basal neuronal properties but specifically inhibited synaptic plasticity, which is regulated by NFIA in astrocytes through calcium-dependent mechanisms. Together, our studies reveal region-specific transcriptional dependencies for astrocytes and identify astrocytic NFIA as a key transcriptional regulator of hippocampal circuits.

Novel phospho-switch function of delta-catenin in dendrite development.

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development.

Caveolin-1-mediated sphingolipid oncometabolism underlies a metabolic vulnerability of prostate cancer.

Plasma and tumor caveolin-1 (Cav-1) are linked with disease progression in prostate cancer. Here we report that metabolomic profiling of longitudinal plasmas from a prospective cohort of 491 active surveillance (AS) participants indicates prominent elevations in plasma sphingolipids in AS progressors that, together with plasma Cav-1, yield a prognostic signature for disease progression. Mechanistic studies of the underlying tumor supportive onco-metabolism reveal coordinated activities through which Cav-1 enables rewiring of cancer cell lipid metabolism towards a program of 1) exogenous sphingolipid scavenging independent of cholesterol, 2) increased cancer cell catabolism of sphingomyelins to ceramide derivatives and 3) altered ceramide metabolism that results in increased glycosphingolipid synthesis and efflux of Cav-1-sphingolipid particles containing mitochondrial proteins and lipids. We also demonstrate, using a prostate cancer syngeneic RM-9 mouse model and established cell lines, that this Cav-1-sphingolipid program evidences a metabolic vulnerability that is targetable to induce lethal mitophagy as an anti-tumor therapy.

REST-DRD2 mechanism impacts glioblastoma stem cell-mediated tumorigenesis.

BACKGROUND: Glioblastoma (GBM) is a lethal, heterogeneous human brain tumor, with regulatory mechanisms that have yet to be fully characterized. Previous studies have indicated that the transcriptional repressor REST (repressor element-1 silencing transcription factor) regulates the oncogenic potential of GBM stem cells (GSCs) based on level of expression. However, how REST performs its regulatory role is not well understood. METHODS: We examined 2 independent high REST (HR) GSC lines using genome-wide assays, biochemical validations, gene knockdown analysis, and mouse tumor models. We analyzed in-house patient tumors and patient data present in The Cancer Genome Atlas (TCGA). RESULTS: Genome-wide transcriptome and DNA-binding analyses suggested the dopamine receptor D2 (DRD2) gene, a dominant regulator of neurotransmitter signaling, as a direct target of REST. Biochemical analyses and mouse intracranial tumor models using knockdown of REST and double knockdown of REST and DRD2 validated this target and suggested that DRD2 is a downstream target of REST regulating tumorigenesis, at least in part, through controlling invasion and apoptosis. Further, TCGA GBM data support the presence of the REST-DRD2 axis and reveal that high REST/low DRD2 (HRLD) and low REST/high DRD2 (LRHD) tumors are specific subtypes, are molecularly different from the known GBM subtypes, and represent functional groups with distinctive patterns of enrichment of gene sets and biological pathways. The inverse HRLD/LRHD expression pattern is also seen in in-house GBM tumors. CONCLUSIONS: These findings suggest that REST regulates neurotransmitter signaling pathways through DRD2 in HR-GSCs to impact tumorigenesis. They further suggest that the REST-DRD2 mechanism forms distinct subtypes of GBM.